Acetylcholinesterase is a serine hydrolase whose primary function is the hydrolysis of acetylcholine at cholinergic synapses in many animals. As a result of a gene duplication early in vertebrate evolution, humans and other vertebrates have another cholinesterase, butyrylcholinesterase, which appears to act as a scavenger of a variety of toxins in the body. Cholinesterases are of considerable physiological, pharmacological, toxicological, and insecticidal interest. Four of the five FDA-approved treatments for Alzheimer's disease are acetylcholinesterase inhibitors. Reprehensibly, the function of acetylcholinesterase in humans also makes it the target of organophosphate nerve gases. The ubiquity of cholinergic synapses in the animal kingdom has also led to the development of numerous organophosphate and carbamate acetylcholinesterase inhibitors as insecticides. The known structures of acetylcholinesterase and butyrylcholinesterase, molecular modeling, site-directed mutagenesis, and in vitro expression of acetylcholinesterase and butyrylcholinesterase from various species have allowed a comparative biochemical approach that has identified residues comprising important catalytic subsites in the enzymes: the catalytic triad, the hydrophobic patch, the acyl pocket, the oxyanion hole, and the peripheral site. We propose to extend this comparative biochemical and molecular biological approach that has worked so well for vertebrate acetylcholinesterase and butyrylcholinesterase to include cholinesterase 1 and cholinesterase 2 from the invertebrate chordate amphioxus, an organism that occupies a key position in evolution, and that has cholinesterases with unique properties. This approach has already been used profitably for cholinesterase 2 and should continue to provide additional insights into the structure and function of cholinesterases, revealing fundamental catalytic requirements, and perhaps leading to the design of more specific, effective, and safer pharmaceuticals and pesticides.